This proposal is designed to address questions concerning the mechanism of escape of the intracellular pathogen Listeria monocytogenes (LM), from the host cell's endosomal/lysosomal degradative pathway. The Gram-positive LM belongs to a class of facultative intracellular pathogens that are capable of growing and replicating within the cytosol of host cells. The ability of LM to escape from the endocytotic vacuole is due primarily to the presence of secreted listeriolysin O (LLO), and possibly to the presence of secreted phospholipases whose exact roles are unknown. Binding of LLO to cholesterol is postulated to be followed by membrane insertion and homo-oligomerization to form pores. Pore formation is thought to be at least partly responsible for the escape of LM into the cytosol, although the exact molecular nature and size of the pore are not completely understood. LLO is secreted as a water-soluble monomer that apparently undergoes a conformational change at a pH optimum of 5.5-5.9, resulting in the protein's ability to insert into lipid membranes. However, a complete understanding of the biochemical and biophysical events that account for the transition from a water-soluble state to one favoring the protein's presence in a hydrophobic environment is currently lacking. LLO has several features, particularly its low optimal pH, that make it a favorable candidate as a vehicle for cytosolic drug delivery. We plan to exploit these attributes of LLO, in combination with liposomes, to increase the efficiency of the delivery of therapeutic macromolecules such as plasmid DNA to the cytosol of mammalian cells.